Diagnosis and treatment of tumor-suppressor associated disorders

ABSTRACT

Methods are provided for detecting a cell proliferative disorder associated with TSLC1 by contacting a proliferating cell of a subject suspected of having the disorder with a reagent that detects TSLC1 and detecting the level of TSLC1 in the proliferating cell. TSLC1 is a single gene whose expression is reduced or absent in A549 and some other NSCLC, hepatocellular carcinoma and pancreatic cancer cell lines. It has further been discovered that TSLC1 expression or suppression is perfectly correlated with promoter methylation state. Restoration of TSLC1 expression to normal or higher levels is sufficient by itself to suppress tumor formation. The invention further provides methods of treating such disorders by contacting cells of a patient suffering from the disorder with a therapeutically effective amount of a reagent that modulates TSLC1 level in the proliferating cells.

This application claims priority under 35 U.S.C. §119(e) of U.S.Provisional Application Serial No. 60/225,264, filed Aug. 15, 2000, theentire contents of which is incorporated herein by reference.

This invention was made in part with government support under Grant No.2P01HD24605 awarded by the National Institutes of Health and U.S. PublicHealth Service award HD-24605. The government may have certain rights inthis invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to screening assays and molecularmedicine, and more specifically to methods for identifying individualshaving or at risk of developing cancer, for grading the severity anddetermining the prognosis of such cancers, and for treating orpreventing such cancers.

2. Background Information

Lung cancer is the leading cause of cancer death, and 80% of lungcancers are non-small cell lung cancer (NSCLC). While human lung canceris not thought of as a genetic disease, a variety of molecular geneticstudies have shown that lung cancer cells have acquired a number ofgenetic lesions including activation of dominant oncogenes andinactivation of tumor suppressor or recessive oncogenes. In fact, itappears that to become clinically evident, lung cancer cells have toaccumulate a large number (perhaps 10 or more) of such lesions. For thedominant oncogenes, these include point mutations in the coding regionsof the ras family of oncogenes (particularly in the K-ras gene inadenocarcinoma of the lung) and amplification, rearrangements, and/orloss of transcriptional control of myc family oncogenes (c-, N-, andL-myc), with changes in c-myc found in non-small cell cancers whilechanges in all myc family members are found in small cell lung cancer.Tumor mutations in ras genes are associated with poor prognosis innon-small cell lung cancer, while tumor amplification of c-myc isassociated with poor prognosis in small cell lung cancers.

For the recessive oncogenes (tumor suppressor genes), cytogenetic andrestriction fragment length polymorphism (RFLP) analyses have showndeletions (allele loss) involving chromosome regions 1p, 1q, 3p14, 3p21,3p24-25, 3q, 5q (familial polyposis gene cluster), 9p (interferon genecluster), 11p, 13q14 (retinoblastoma, rb, gene) 16q, and 17p13 (p53gene), as well as other sites. There appear to be several candidaterecessive oncogenes on chromosome 11q that are involved in nearly alllung cancers.

The large number of genetic lesions in clinically evident lung cancerhas prompted a search for these mutations in lung tissue before classiccytopathologic evidence of malignancy can be found, to provide formolecular early diagnosis and as intermediate endpoints in preventionefforts, including chemo-prevention treatment.

Pancreatic cancer is the fourth leading cause of cancer death in men andin women and each year ˜28,000 Americans die of the disease (6).Frequent genetic changes such as mutational activation of the K-rasoncogene and inactivation of the p 16, DPC4, p53, MKK4, STK11, TGFBR2,and TGFBR1 tumor suppressor genes have been described in pancreaticcancer (7, 8). Although multiple tumor suppressor pathways have beenshown to play a role in pancreatic carcinogenesis, little is known aboutthe contribution of DNA methylation to inactivation of genes in thesepathways. Recently, a novel technique, methylated CpG islandamplification (MCA), was developed to enrich for methylated CpG richsequences. MCA coupled with RDA (MCA/RDA) can recover CpG islandsdifferentially methylated in cancer cells.

Primary hepatocellular carcinoma is one of the most common tumors in theworld. It is especially prevalent in regions of Asia and sub-SaharanAfrica, where the annual incidence is up to 500 cases per 1000,000population. In the United States and western Europe, it is much lesscommon, accounting for only 1 to 2 percent of malignant tumors atautopsy. Hepatocellular carcinoma is up to four times for common in menthan in women and usually arises in a cirrhotic liver.

The principal reason for the high incidence of hepatocellular carcinomain parts of Asia and Africa is the frequency of chronic infection withhepatitis B virus (HBV) and hepatitis C virus (HBC). These chronicinfections frequently lead to chirrhosis, which itself is an importantrisk factor for hepatocellular cancinomas. In patients with HBVinfection and hepatocellular carcinoma, there can be modifications ofcellular gene expression by insertional mutagenesis, chromosomalrearrangements, or the transcriptional transactivating activity of the Xand the pre-52/S regions of the HBV genome. These alternations probablyoccur during the process of liver cell injury and repair.

Thus, there is a need in the art for new and better methods fordiagnosing individuals having or at risk of developing lung, liver andpancreatic cancers as well as a need for methods of treatment of suchconditions.

SUMMARY OF THE INVENTION

The present invention is based on the seminal discovery that a region of700 kb on 11q23.2 can suppress tumorigenicity of A549 human non-smallcell lung cancer (NSCLC) cells, as well as some other NSCLC,hepatocellular carcinoma (HCC) and pancreatic cancer (PAC) cell lines.Accordingly, the present invention provides methods of detecting a cellproliferative disorder associated with tumor suppressor lung cancer 1(TSLC1) in a subject in need thereof by contacting a cell component of aproliferating cell of the subject with a reagent that detects the levelof the cell component in the proliferating cell and determining amodification in the level of the cell component in the proliferatingcell as compared with a comparable healthy cell, wherein the cellcomponent indicates the level of TSLC1 in the cell and the modificationindicates the disorder associated with TSLC1.

In another embodiment, the present invention provides methods ofdetecting a cell proliferative disorder in a subject in need thereof bycontacting a target cellular component of a test cell with a reagentthat detects the level of TSLC1 and detecting a reduction in the levelof TSLC1 in the proliferating cell as compared to that of a comparablenormal cell; wherein the cell proliferative disorder is aTSLC1-associated lung, liver or pancreatic cancer.

In yet another embodiment, the present invention provides methods oftreating a cell proliferative disorder associated with modification ofTSLC1 production in proliferating cells in a subject in need thereof. Inthe invention therapeutic methods, cells of a patient suffering fromsuch a disorder are contacted with a therapeutically effective amount ofa reagent that modulates TSLC1 level in the proliferating cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of DNA segments used forfunctional mapping of tumor suppressor activity of TSLC1 using 1.6 MBYAC and nested deletion derivatives of it transfected into A549 NSCLCtumor cells. TSLC1 was localized to a 100 kb segment responsibleessential for full suppression. SHGC-31226 is an EST clone mapped ony939b12. (B) Variable expression of TSLC1 in human tissues.

FIG. 1B is a graph showing the average volume of tumors that formed ateight sites as determined at the indicated times after injection of 10⁵cells into live nude mice from the following A549 derivatives: A549transfected with control plasmid (); ATSLC1 (◯), ATSLC2 (▪), ATSLC3(Δ), AΔTSLC (□).

FIG. 2A shows a comparison the sequence of exon 10 of TSLC1 in L255N andL255C and identifies a 2 bp deletion (underlined) in L255C.

FIG. 2B shows a comparison of the sequence of exon 5 of TSLC1 in H13NAND H13C and illustrates that codon 208 (underlined ) is mutated in theTSLC1 gene in H13C.

FIG. 2C shows the sequence of a CpG island upstream from TSLC1 (SEQ IDNO:24) (with nucleic acids 62-79 boxed). Primer sequences used toamplify this region are underlined and CpG sites, number 1-6, are doublyunderlined. Predicted TATA box sequence is shown with a dashedunderline.

FIG. 2D shows residues 62-79 bp of the 93-bp fragment of the CpG islandin normal lung, (SEQ ID NO:25), SK-LU-1 (SEQ ID NO:26), Calu-3 (SEQ IDNO:27), and A549 (SEQ ID NO:28). Bisulfite sequencing identified threemethylated cytosine residues in SK-LU-1 and Calu-3 cell lines, but notin A549 or normal lung. Asterisks indicate the nucleotides correspondingto methylated cytosine residues at CpG sites.

FIG. 2E is a chart showing the methylation status of the TSLC1 promoterin a normal lung and 12 NSCLC cell lines. White and black circlesrepresent unmethylated and methylated CpGs, respectively. Grey circlesrepresent partially methylated CpGs. Columns correspond to the sixidentified sites of methylated cytosine residues in CpG sites shown inFIG. 2D.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the seminal discovery that a region of700 kb on 11q23.2 can suppress tumorigenicity of A549 human non-smallcell lung cancer (NSCLC) cells, as well as some other NSCLC,hepatocellular carcinoma (HCC) and pancreatic cancer (PAC) cell lines.Most of this tumor suppressor activity localizes to a 100 kb segment.This region contains a single gene, TSLC1 (bases 25 through 1353 of apolynucleotide available as DNA Data Base of Japan (DDBJ) Accession No.AB017563) (SEQ ID NO:2), whose expression is reduced or absent in A549and some other NSCLC, hepatocellular carcinoma and pancreatic cancercell lines. It has further been discovered that TSLC1 expression orsuppression is perfectly correlated with promoter methylation state.Restoration of TSLC1 expression to normal or higher levels is sufficientby itself to suppress tumor formation by A549 cells in nude mice. Theseresults, and the identification of truncating mutations uncovered byloss of the wild type allele detected in a primary lung and a livertumor, suggest that attenuation of TSLC1 expression is involved inmultiple human cancers.

Accordingly, the present invention provides methods of detecting a cellproliferative disorder associated with tumor suppressor lung cancer 1(TSLC1) in a subject in need thereof by contacting a cell component of aproliferating cell of the subject with a reagent that detects the levelof the cell component in the proliferating cell and determining amodification in the level of the cell component in the proliferatingcell as compared with a comparable healthy cell, wherein the cellcomponent indicates the level of TSLC1 in the cell and the modificationindicates the disorder associated with TSLC1. The target cell componentcontacted can be nucleic acid, such as DNA or RNA, or it can be protein.When the component is nucleic acid, the reagent is typically a nucleicacid probe or PCR primer. When the cell component is protein, thereagent is typically an anti-TSLC1 antibody probe. The target cellcomponent may be detected directly in situ or it may be isolated fromother cell components by common methods known to those of skill in theart before contacting with a probe. (See for example, Maniatis, et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y., 1989; Current Protocols in Molecular Biology, 1994, Ed. Ausubel,et al., Greene Publ. Assoc. & Wiley Interscience.)

Detection methods include Southern and Northern blot analyses, RNaseprotection, immunoassays and other detection assays that are known tothose of skill in the art.

The probes can be detectably labeled, for example, with a radioisotope,a fluorescent compound, a bioluminescent compound, a chemiluminescentcompound, a metal chelator, or an enzyme. Those of ordinary skill in theart will know of other suitable labels for binding to the probes or willbe able to ascertain such, using routine experimentation.

Since the present invention shows that a decreased level of TSLC1transcription is often the result of hypermethylation of the TSLC1 gene,it is often desirable to directly determine whether the TSLC1 gene ishypermethylated. In particular, the cytosine rich areas termed “CpGislands,” which lie in the 5′ regulatory regions of genes are normallyunmethylated. The term “hypermethylation” includes any methylation ofcytosine at a position that is normally unmethylated in the TSLC1 genesequence (e.g. the TSLC1 promoter). Hypermethylation can be detected byrestriction endonuclease treatment of TSLC1 polynucleotide (gene) andSouthern blot analysis for example. Therefore, in an invention methodwherein the cellular component detected is DNA, restriction endonucleaseanalysis is preferable to detect hypermethylation of the TSLC1 gene. Anyrestriction endonuclease that includes CG as part of its recognitionsite and that is inhibited when the C is methylated, can be utilized.Methylation sensitive restriction endonucleases such as BssHII, MspI,NotI or HpaII, used alone or in combination, are examples of suchendonucleases. Other methylation sensitive restriction endonucleaseswill be known to those of skill in the art. In addition, PCR can beutilized to detect the methylation status of the TSLC1 gene.Oligonucleotide primers based on any coding sequence region in the TSLC1sequence are useful for amplyifying DNA by PCR in the invention methods.

For purposes of the invention, an antibody (i.e., an anti-TSLC1antibody) or nucleic acid probe specific for TSLC1 may be used to detectthe presence of TSLC1 polypeptide (using antibody) or polynucleotide(using nucleic acid probe) in biological fluids or tissues.Oligonucleotide primers based on any coding sequence region in the TSLC1sequence are useful for amplifying DNA, for example by PCR. Any specimencontaining a detectable amount of TSLC1 polynucleotide or TSLC1polypeptide antigen can be used. Nucleic acid can also be analyzed byRNA in situ methods that are known to those of skill in the art andillustrated in the Examples contained herein. Preferred tissues fortesting or treating according to the invention methods are tissue oflung, pancreas, liver, and the like. Although the subject can be anymammal, preferably the subject is human.

Various disorders that are detectable by the method of the inventioninclude non-small cell lung cancer, hepatocellular carcinoma, pancreaticcancer, and the like.

The invention methods can utilize antibodies immunoreactive with TSLC1polypeptide (SEQ ID NO:1) (a predicted amino acid sequence available asGenBak Accession No. BAA75822) or immunoreactive fragments thereof.Antibody that consists essentially of pooled monoclonal antibodies withdifferent epitopic specificities, as well as distinct monoclonalantibody preparations can be used. Monoclonal antibodies are made fromantigen containing fragments of the protein by methods well known tothose skilled in the art (Kohler, et al., Nature, 256:495, 1975). Theterm antibody as used in this invention is meant to include intactmolecules as well as fragments thereof, such as Fab and F(ab′)₂, whichare capable of binding an epitopic determinant on TSLC1.

Monoclonal antibodies can be used in the invention diagnostic methods,for example, in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier. In addition, the monoclonalantibodies in these immunoassays can be detectably labeled in variousways. Examples of types of immunoassays that can utilize monoclonalantibodies of the invention are competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of the antigens using the monoclonalantibodies of the invention can be done utilizing immunoassays that arerun in either the forward, reverse, or simultaneous modes, includingimmunohistochemical assays on physiological samples. Those of skill inthe art will know, or can readily discern, other immunoassay formatswithout undue experimentation.

The term “immunometric assay” or “sandwich immunoassay”, includessimultaneous sandwich, forward sandwich and reverse sandwichimmunoassays. These terms are well understood by those skilled in theart. Those of skill will also appreciate that antibodies according tothe present invention will be useful in other variations and forms ofassays that are presently known or which may be developed in the future.These are intended to be included within the scope of the presentinvention.

Monoclonal antibodies can be bound to many different carriers and usedto detect the presence of TSLC1. Examples of well-known carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding monoclonal antibodies, or will beable to ascertain such using routine experimentation.

In performing the assays it may be desirable to include certain“blockers” in the incubation medium (usually added with the labeledsoluble antibody). The “blockers” are added to assure that non-specificproteins, proteases, or anti-heterophilic immunoglobulins to anti-TSLC1immunoglobulins present in the experimental sample do not cross-link ordestroy the antibodies on the solid phase support, or the radiolabeledindicator antibody, to yield false positive or false negative results.The selection of “blockers” therefore may add substantially to thespecificity of the assays described in the present invention.

It has been found that a number of nonrelevant (i.e., nonspecific)antibodies of the same class or subclass (isotype) as those used in theassays (e.g., IgG1, IgG2a, IgM, etc.) can be used as “blockers”. Theconcentration of the “blockers” (normally 1-100 μg/μl) may be important,in order to maintain the proper sensitivity yet inhibit any unwantedinterference by mutually occurring cross-reactive proteins in thespecimen.

In using a monoclonal antibody for the in vivo detection of antigen, thedetectably labeled monoclonal antibody is given in a dose that isdiagnostically effective. The term “diagnostically effective” means thatthe amount of detectably labeled monoclonal antibody is administered insufficient quantity to enable detection of the site having the TSLC1antigen for which the monoclonal antibodies are specific. Theconcentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cellshaving TSLC1 is detectable compared to the background, depending uponthe in vivo imaging or detection method employed, such as MRI, CAT scan,and the like. Further, it is desirable that the detectably labeledmonoclonal antibody be rapidly cleared from the circulatory system inorder to give the best target-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. The dosage of monoclonal antibodycan vary from about 0.001 mg/m² to about 500 mg/m², preferably 0.1 mg/m²to about 200 mg/m², most preferably about 0.1 mg/m² to about 10 mg/m².Such dosages may vary, for example, depending on whether multipleinjections are given, tumor burden, and other factors known to those ofskill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay that is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

For in vivo diagnosis, radioisotopes can be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions that can be bound to the monoclonalantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and²⁰¹T1.

A monoclonal antibody useful in the invention methods can also belabeled with a paramagnetic isotope for purposes of in vivo diagnosis,as in magnetic resonance imaging (MRI) or electron spin resonance (ESR).In general, any conventional method for visualizing diagnostic imagingcan be utilized. Usually gamma and positron emitting radioisotopes areused for camera imaging and paramagnetic isotopes for MRI. Elements thatare particularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy,⁵²Cr, and ⁵⁶Fe.

The present invention also provides methods for treating a subject witha cell proliferative disorder associated with TSLC1 comprisingadministering to a subject with the disorder a therapeutically effectiveamount of a reagent that modulates TSLC1 expression. In non-small lungcancer, hepatocellular carcinoma and pancreatic cancer cells, forexample, the TSLC1 nucleotide sequence is under-expressed as compared toexpression in a normal cell, therefore, it is possible to designappropriate therapeutic or diagnostic techniques directed to thissequence. Thus, where a cell-proliferative disorder is associated withthe expression of TSLC1 associated with malignancy, nucleic acidsequences that modulate TSLC1 expression at the transcriptional ortranslational level can be used. In cases when a cell proliferativedisorder or abnormal cell phenotype is associated with the underexpression of TSLC1, for example, nucleic acid sequences encoding TSLC1(sense) could be administered to the subject with the disorder.

The term “cell-proliferative disorder” denotes malignant as well asnon-malignant cell populations, which often appear to differ from thesurrounding tissue both morphologically and genotypically. Suchdisorders may be associated, for example, with absence of or reducedexpression of TSLC1. Essentially, any disorder that is etiologicallylinked to expression of TSLC1 could be considered susceptible totreatment using invention methods that employ a reagent of the inventionto modulate TSLC1 expression.

The term “modulate” encompasses the suppression of methylation of TSLC1polynucleotide when TSLC1 is under-expressed. When a cell proliferativedisorder is associated with TSLC1 expression, such methylationsuppressive reagents as 5-azacytadine can be introduced to a cell.Alternatively, when a cell proliferative disorder is associated withunder-expression of TSLC1 polypeptide, a sense polynucleotide sequence(the DNA coding strand) encoding TSLC1 polypeptide, or 5′ regulatorynucleotide sequences (i.e., promoter) of TSLC1 in operable linkage withTSLC1 polynucleotide can be introduced into the cell. Demethylases knownin the art could also be used to remove methylation.

The present invention also provides gene therapy for the treatment ofcell proliferative disorders that are mediated by TSLC1. Such therapywould achieve its therapeutic effect by introduction of the appropriateTSLC1 polynucleotide that contains a TSLC1 structural gene (sense), intocells of subjects having the proliferative disorder. Delivery of senseTSLC1 polynucleotide constructs can be achieved using a recombinantexpression vector such as a chimeric virus or a colloidal dispersionsystem.

The polynucleotide sequences used in the methods of the invention may bethe native, unmethylated sequence or, alternatively, may be a sequencein which a nonmethylatable analog is substituted within the sequence.Preferably, the analog is a nonmethylatable analog of cytidine, such as5-azacytadine. Other analogs will be known to those of skill in the art.Alternatively, such nonmethylatable analogs could be administered to asubject as drug therapy, alone or simultaneously with a sense structuralgene for TSLC1 or sense promoter for TSLC1 operably linked to TSLC1structural gene.

In another embodiment, a TSLC1 structural gene is operably linked to atissue specific heterologous promoter and used for gene therapy. Forexample, a TSLC1 gene can be ligated to hepatocellular-specific promoterfor expression of TSLC1 in hepatocellular tissue. Other tissue specificpromoters will be known to those of skill in the art. Alternatively, thepromoter for another tumor suppressor gene can be linked to the TSLC1structural gene and used for gene therapy.

Various viral vectors that can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). Most preferably, a non-human primate retroviralvector is employed, such as the gibbon ape leukemia virus (GaLV),thereby providing a broader host range than murine vectors, for example.

A number of additional retroviral vectors can incorporate multiplegenes. All of these vectors can transfer or incorporate a gene for aselectable marker so that transduced cells can be identified andgenerated. Retroviral vectors can be made target specific by inserting,for example, a polynucleotide encoding a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody totarget the retroviral vector. Those of skill in the art will know of, orcan readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome to allow target specific delivery of the retroviral vectorcontaining the TSLC1 sense or antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence that enables the packaging mechanism to recognize anRNA transcript for encapsidation. Helper cell lines that have deletionsof the packaging signal include but are not limited to PS12, PA317 andPA12, for example. These cell lines produce empty virions, since nogenome is packaged. If a retroviral vector is introduced into such cellsin which the packaging signal is intact, but the structural genes arereplaced by other genes of interest, the vector can be packaged andvector virion produced.

Another targeted delivery system for TSLC1 polynucleotide is a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. The preferred colloidal system of this invention is aliposome. Liposomes are artificial membrane vesicles that are useful asdelivery vehicles in vitro and in vivo. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2-4.0 um, canencapsulate a substantial percentage of aqueous buffer containing largemacromolecules. RNA, DNA and intact virions can be encapsulated withinthe aqueous interior and be delivered to cells in a biologically activeform (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition tomammalian cells, liposomes have been used for delivery ofpolynucleotides in plant, yeast and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genes ofinterest at high efficiency while not compromising their biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information (Mannino, etal., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes has been classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal-targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

In general, the compounds bound to the surface of the targeted deliverysystem will be ligands and receptors which will allow the targeteddelivery system to find and “home in” on the desired cells. A ligand maybe any compound of interest that will bind to another compound, such asa receptor.

In general, surface membrane proteins that bind to specific effectormolecules are referred to as receptors. In the present invention,antibodies are preferred receptors. Antibodies can be used to targetliposomes to specific cell-surface ligands. For example, certainantigens expressed specifically on tumor cells, referred to astumor-associated antigens (TAAs), may be exploited for the purpose oftargeting TSLC1 antibody-containing liposomes directly to the malignanttumor. Since the TSLC1 gene product may be indiscriminate with respectto cell type in its action, a targeted delivery system offers asignificant improvement over randomly injecting non-specific liposomes.Preferably, the target tissue is human brain, colon, breast, lung, andrenal origin. A number of procedures can be used to covalently attacheither polyclonal or monoclonal antibodies to a liposome bilayer.Antibody-targeted liposomes can include monoclonal or polyclonalantibodies or fragments thereof such as Fab, or F(ab′)₂, as long as theybind efficiently to an antigenic epitope on the target cells. Liposomesmay also be targeted to cells expressing receptors for hormones or otherserum factors.

For use in the diagnostic research and therapeutic applicationssuggested above, kits are also provided by the invention. Such a kit maycomprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method.

For example, one of the container means may comprise a probe that is orcan be detectably labeled. Such probe may be an antibody or nucleotidespecific for a target protein or a target nucleic acid, respectively,wherein the target is indicative, or correlates with, the presence ofTSLC1 of the invention. Where the kit utilizes nucleic acidhybridization to detect the target nucleic acid, the kit may also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradionucleotide label.

The invention methods utilize a functional polypeptide, TSLC1, andfunctional fragments thereof. As used herein, the term “functionalpolypeptide” refers to a polypeptide that possesses a biologicalfunction or activity which is identified through a defined functionalassay and which is associated with a particular biologic, morphologic,or phenotypic alteration in the cell. Functional fragments of the TSLC1polypeptide include fragments of TSLC1 that retain the activity of e.g.,tumor suppressor activity, of TSLC1. Smaller peptides containing thebiological activity of TSLC1 are included in the invention. Thebiological function, for example, can vary from a polypeptide fragmentas small as an epitope to which an antibody molecule can bind to a largepolypeptide that is capable of participating in the characteristicinduction or programming of phenotypic changes within a cell.

The invention methods can also utilize a “functional polynucleotide”denotes a polynucleotide which encodes a functional TSLC1 polypeptide asdescribed herein.

Minor modifications of the TSLC1 primary amino acid sequence may resultin proteins that have substantially equivalent activity as compared tothe TSLC1 polypeptide described herein. Such modifications may bedeliberate, as by site-directed mutagenesis, or may be spontaneous. Allof the polypeptides produced by these modifications are included hereinas long as the tumor suppressor activity of TSLC1 is present. Further,deletion of one or more amino acids can also result in a modification ofthe structure of the resultant molecule without significantly alteringits activity. This can lead to the development of a smaller activemolecule that would have broader utility. For example, it possible toremove amino or carboxy terminal amino acids that may not be requiredfor TSLC1 activity.

The TSLC1 polypeptide used in the invention methods or encoded bypolynucleotides used in the invention methods also includes conservativevariations of the polypeptide sequence. The term “conservativevariation” as used herein denotes the replacement of an amino acidresidue by another, biologically similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acids, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

The invention methods also can utilize an isolated polynucleotidesequence consisting essentially of a polynucleotide sequence encoding apolypeptide having the amino acid sequence of SEQ ID NO:1. Thepolynucleotide sequence may also includes the 5′ and 3′ untranslatedsequences and regulatory sequences, for example. The term “isolated” asused herein includes polynucleotides substantially free of other nucleicacids, proteins, lipids, carbohydrates or other materials with which itis naturally associated. Polynucleotide sequences of the inventioninclude DNA, cDNA and RNA sequences that encode TSLC1. It is understoodthat all polynucleotides encoding all or a portion of TSLC1 can also beused in the invention methods, as long as they encode a polypeptide withTSLC1 activity. For example, the polynucleotide can be a nucleic acidprobe having a nucleotide sequence a) as set forth in nucleic acidresidues 411-1,371 of cDNA encoding TSLC1 (SEQ ID NO:17), b) apolynucleotide having at least 70% identity or complementary to such apolynucleotide, or a polynucleotide comprising at least 15 bases of apolynucleotide of a) or b).

Such polynucleotides include naturally occurring, synthetic, andintentionally manipulated polynucleotides. For example, TSLC1polynucleotide may be subjected to site-directed mutagenesis. Thepolynucleotide sequence for TSLC1 also includes antisense sequences. Thepolynucleotides of the invention include sequences that are degenerateas a result of the genetic code. There are 20 natural amino acids, mostof which are specified by more than one codon. Therefore, all degeneratenucleotide sequences are included in the invention as long as the aminoacid sequence of TSLC1 polypeptide encoded by the nucleotide sequence isfunctionally unchanged. In addition, the invention also includes apolynucleotide consisting essentially of a polynucleotide sequenceencoding a polypeptide having an amino acid sequence of SEQ ID NO:3 andhaving at least one epitope for an antibody immunoreactive with TSLC1polypeptide.

“Identity” refers to sequence similarity between two peptides or betweentwo nucleic acid molecules. Homology can be determined by comparing aposition in each sequence that may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences.

The term “transfection” or “transforming” and grammatical equivalentsthereof, refers to the introduction of a nucleic acid, e.g., anexpression vector, into a recipient cell by nucleic acid-mediated genetransfer. “Transformation”, as used herein, refers to a process in whicha cell's genotype is changed as a result of the cellular uptake ofexogenous DNA or RNA, and, for example, the transformed cell expresses arecombinant form of one of the invention family of TSCL1 tumorsuppressors.

“Cells” or “cell cultures” or “recombinant host cells” or “host cells”are often used interchangeably as will be clear from the context. Theseterms include the immediate subject cell that expresses the tumorsuppressor protein of the present invention, and, of course, the progenythereof. It is understood that not all progeny are exactly identical tothe parental cell, due to chance mutations or difference in environment.However, such altered progeny are included in these terms, so long asthe progeny retain the characteristics relevant to those conferred onthe originally transformed cell. In the present case, such acharacteristic might be the ability to produce a recombinant TSCL1 tumorsuppressor polypeptide.

DNA sequences used in the invention methods can be obtained by severalmethods. For example, the DNA can be isolated using hybridizationtechniques that are well known in the art. These include, but are notlimited to: 1) hybridization of genomic or cDNA libraries with probes todetect homologous nucleotide sequences and 2) antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features.

Preferably the TSLC1 polynucleotide used in the invention methods isderived from a mammalian organism, and most preferably from human.Oligonucleotide probes, which correspond to a part of the sequenceencoding the protein in question, can be synthesized chemically for usein the invention methods. This requires that short, oligopeptidestretches of amino acid sequence must be known. The DNA sequenceencoding the protein can be deduced from the genetic code; however, thedegeneracy of the code must be taken into account. It is possible toperform a mixed addition reaction when the sequence is degenerate. Thisincludes a heterogeneous mixture of denatured double-stranded DNA. Forsuch screening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. In other words, by using stringenthybridization conditions directed to avoid non-specific binding, it ispossible, for example, to allow the autoradiographic visualization of aspecific cDNA clone by the hybridization of the target DNA to thatsingle probe in the mixture which is its complete complement (Wallace,et al., Nucl. Acid Res., 9:879, 1981).

Specific DNA sequences encoding TSLC1 for use in the invention methodscan also be obtained by: 1) isolation of double-stranded DNA sequencesfrom the genomic DNA; 2) chemical manufacture of a DNA sequence toprovide the necessary codons for the polypeptide of interest; and 3) invitro synthesis of a double-stranded DNA sequence by reversetranscription of mRNA isolated from a eukaryotic donor cell. In thelatter case, a double-stranded DNA complement of MRNA is eventuallyformed which is generally referred to as cDNA.

Of the three above-noted methods for developing specific DNA sequencesfor use in recombinant procedures, the isolation of genomic DNA isolatesis the least common. This is especially true when it is desirable toobtain the microbial expression of mammalian polypeptides due to thepresence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librariesthat are derived from reverse transcription of mRNA that is abundant indonor cells that have a high level of gene expression. When used incombination with polymerase chain reaction technology, even rareexpression products can be cloned, as is illustrated in the Examplesherein. In those cases where significant portions of the amino acidsequence of the polypeptide are known, the production of labeled singleor double-stranded DNA or RNA sequences duplicating a sequenceputatively present in the target cDNA may be employed in DNA/DNAhybridization procedures which are carried out on cloned copies of thecDNA which have been denatured into a single-stranded form (Jay, et al.,Nucl. Acid Res., 11:2325, 1983).

A cDNA expression library, such as lambda gt11, can be screenedindirectly to obtain TSLC1 peptides having at least one epitope, usingantibodies specific for TSLC1. Such antibodies can be eitherpolyclonally or monoclonally derived and used to detect expressionproduct indicative of the presence of TSLC1 cDNA in a test suspected ofbeing a proliferating cell.

DNA sequences encoding TSLC1 can be expressed in vitro by DNA transferinto a suitable host cell, such as a tumor cell. “Host cells” are cellsin which a vector can be propagated and its DNA expressed. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used. Methods of stable transfer,meaning that the foreign DNA is continuously maintained in the host, areknown in the art.

In the present invention, the TSLC1 polynucleotide sequences may beinserted into a recombinant expression vector for expression either invivo or in vitro. The term “recombinant expression vector” refers to aplasmid, virus or other vehicle known in the art that has beenmanipulated by insertion or incorporation of the TSLC1 geneticsequences. Such expression vectors contain a promoter sequence thatfacilitates the efficient transcription of the inserted genetic sequenceof the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific genes that allow phenotypicselection of the transformed cells. Vectors suitable for use in thepresent invention include, but are not limited to the T7-basedexpression vector for expression in bacteria (Rosenberg, et al., Gene56:125, 1987), the pMSXND expression vector for expression in mammaliancells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

Polynucleotide sequences encoding TSLC1 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention.

Methods that are well known to those skilled in the art can be used toconstruct expression vectors containing the TSLC1 coding sequence andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo recombination/genetic techniques. See, for example, thetechniques described in Maniatis, et al., 1989 Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y.

A variety of host-expression vector systems may be utilized to expressthe TSLC1 coding sequence. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the TSLC1 coding sequence; yeast transformed with recombinantyeast expression vectors containing the TSLC1 coding sequence; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the TSLC1 coding sequence; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing the TSLC1 coding sequence; or animal cell systems infectedwith recombinant virus expression vectors (e.g., retroviruses,adenovirus, vaccinia virus) containing the TSLC1 coding sequence, ortransformed animal cell systems engineered for stable expression. SinceTSLC1 has not been confirmed to contain carbohydrates, both bacterialexpression systems as well as those that provide for translational andpost-translational modifications may be used; e.g., mammalian, insect,yeast or plant expression systems.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, transcription enhancer elements, transcriptionterminators, etc. may be used in the expression vector (see e.g.,Bitter, et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage γ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and thelike may be used. When cloning in mammalian cell systems, promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the retrovirus long terminalrepeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter)may be used. Promoters produced by recombinant DNA or synthetictechniques may also be used to provide for transcription of the insertedTSLC1 coding sequence. In addition, the endogenous TSLC1 promoter, or amutation thereof to protect the promoter from hypermethylation, may alsobe used to provide transcription machinery of TSLC1.

When the expression vector is introduced into a mammalian host cell inpractice of the invention methods, a eukaryotic systems, and preferablymammalian expression systems, allows for proper post-translationalmodifications of expressed mammalian proteins to occur. Eukaryotic cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, phosphorylation, and advantageously,secretion of the gene product may be used as host cells for theexpression of TSLC1.

Recombinant viruses or viral elements may be used to direct expressionin mammalian cells. For example, when using adenovirus expressionvectors, the TSLC1 coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination.

Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the protein in infected host cells (e.g., see Logan & Shenk,Proc. Natl. Acad. Sci. USA, 81:3655-3659, 1984). Alternatively, thevaccinia virus 7.5K promoter may be used (e.g., see, Mackett, et al.,1982, Proc. Natl. Acad. Sci. USA 79:7415-7419; Mackett, et al., J.Virol. 49:857-864, 1984; Panicali, et al., Proc. Natl. Acad. Sci. USA79:4927-4931, 1982). Of particular interest are vectors based on bovinepapilloma virus which have the ability to replicate as extrachromosomalelements (Sarver, et al., Mol. Cell. Biol. 1: 486, 1981). Shortly afterentry of this DNA into mouse cells, the plasmid replicates to about 100to 200 copies per cell. Transcription of the inserted cDNA does notrequire integration of the plasmid into the host's chromosome, therebyyielding a high level of expression. These vectors can be used forstable expression by including a selectable marker in the plasmid, suchas, for example, the neo gene. Alternatively, the retroviral genome canbe modified for use as a vector capable of introducing and directing theexpression of the TSLC1 gene in mammalian host cells (Cone & Mulligan,Proc. Natl. Acad. Sci. USA 81:6349-6353, 1984.

For long-term, high-yield production of recombinant proteins, as in genetherapy, stable expression is preferred. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with the TSLC1 cDNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and, optionally, a selectablemarker. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci that in turn can becloned and expanded into cell lines. For example, following theintroduction of foreign DNA, engineered cells may be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,Cell, 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026, 1962), andadenine phosphoribosyltransferase (Lowy, et al., Cell, 22: 817, 1980)genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., Natl.Acad. Sci. USA, 77:3567, 1980; O'Hare, et al., Proc. Natl. Acad. Sci.USA, 78: 1527, 1981); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, Proc. Natl. Acad Sci. USA, 78: 2072,1981; neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., J. Mol. Biol, 150:1, 1981); and hygro, which confers resistance tohygromycin (Santerre, et al., Gene, 30:147, 1984) genes. Recently,additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman &Mulligan, Proc. Natl. Acad. Sci. USA, 85:8047, 1988); and ODC (omithinedecarboxylase), which confers resistance to the ornithine decarboxylaseinhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987,In: Current Communications in Molecular Biology, Cold Spring HarborLaboratory, ed.).

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl.sub.2method using procedures well known in the art. Alternatively, MgCl₂ orRbCl can be used. Transformation can also be performed after forming aprotoplast of the host cell if desired.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the TSLC1 of the invention,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman, ed., 1982).

Isolation and purification of microbial or host cell expressedpolypeptide, or fragments thereof, provided by the invention, may becarried out by conventional means including preparative chromatographyand affinity and immunological separations involving monoclonal orpolyclonal antibodies.

TSLC1 expression is ubiquitous in normal adult tissues. However, incultured tumor cells and in primary cancers which exhibithypermethylation of the associated CpG island, TSLC1 expression isreduced or absent. For example, the expression of TSLC1 is absent intumors with CpG island hypermethylation, including lung, colon, breastand brain tumors. This expression pattern is consistent with a tumorsuppressor gene function for TSLC1.

Loss of heterozygosity on the long arm of chromosome 11 has beenreported in NSCLC and other cancers (M. Iizuka et al., Genes Chrom.Cancer 13, 40 (1995); D. Rasio et al., Cancer Res. 55, 3988 (1995); andS. S. Wang et al., Genes Chrom. Cancer 25, 154 (1999)). Linkage studiesof NSCLC are precluded because no hereditary forms are known (Y.Murakami et al., Proc. Natl. Acad. Sci. USA, 95, 8153 (1998);Y. Murakamiet al., Cancer Res. 55, 3389 (1995); S. Thiagalingam et al., NatureGenet. 13, 343 (1996)). Several independent studies have suggested thepresence of multiple tumor suppressor genes in this region, includingthe PPP2R1B gene at 11q23-24 (S. S. Wang et al., Science 282, 284(1998).

In the present invention loss of heterozygosity (LOH) studies werecombined with functional complementation of tumorigenicity by yeastartificial chromosomes (YACs) from normal chromosome 11 to localize thesite of tumor suppressor activity within the 11q23-24 region. Thesuppression of tumorigenicity by this technique is considered to providefunctional evidence of the presence of functional tumor suppressor genesin the segment of chromosome 11 inserted (H. Satoh et al., MolCarcinogen 7, 157 (1993)).

The loss of heterozygosity study identified a 5cM commonly deletedregion on 11q23 chromosome (M. Iizuka et al., supra). Transfer ofoverlapping YAC clones containing normal chromosome 11 into a humannon-small cell lung cancer (NSCLC) cell line, A549, and murine LLC lungcancer cell lines were used to detect aberrations in function of tumorsuppressor genes in chromosome 11 associated with NSCLC. By this methoda potential tumor suppressor gene was localized to the central 700-kbfragment of y939b12, a 1.6 Mb YAC (6,9)(FIG. 1A). Then the tumorsuppressor gene, TSLC1 (tumor suppressor in lung cancer 1, alias ST17),was further localized to a 100-kb candidate region of the YAC by thedetermination that cells transfected with truncated clones containing100 kb of the parental YAC, but not those transfected with a 1,000 kbtruncated derivative, showed strong suppressor activity.

Subsequent analysis of y939b12 identified a gene in the 100-kb candidateregion containing TSLC1. A full length cDNA was constructed (asdescribed in Example 1 herein) and found to be identical to a genepreviously identified as belonging to the immunoglobulin superfamily andnamed IGSF4 by H. Gomyo et al., (Genomics 62, 139 (1999)). The genestructure was determined by comparison with genomic sequence, showingthat TSLC1 spans more than 300-kb. The gene was rendered non-functionalby deletion of 100 kb in the truncated YAC as discussed above. The TSLC1gene encodes a putative membrane glycoprotein of 442 amino acids(GenBank Accession No. BAA75822) (SEQ ID NO:1). TSLC1, the encodedprotein, has an extracellular domain containing 3 immunoglubulin-likeC-2-type fragments, one transmembrane domain, and a short cytoplasmicdomain similar to that of glycophorin C.

Since the predicted amino acid sequence of TSLC1 suggests that it is atransmembrane protein, the subcellular localization of TSLC1 wasexamined by expressing a TSLC1:gfp fusion protein in COS 7 cells, asdescribed in Example 2 herein. TSLC1 localized in perinuclear and plasmamembranes. The corresponding ΔTSLC1:gfp protein construct, lacking thesignal peptide, was expressed in COS 7 cells, but failed to localize inplasma membrane. These findings together with the structural homology inthe extracellular domains of TSLC1 to those of NCAM1, NCAM2 and otherimmunoglobulin superfamily proteins, suggest that TSLC1 might beinvolved in interaction of cells with other cells and/or theextracellular matrix.

Northern blot analysis of mRNAs obtained from a number of healthytissues revealed the two expected TSLC1 transcripts of 4.4 kb and 1.6 kb(FIG. 1B). These mRNAs have been shown to express identical proteins (H.Gomyo et al., Genomics 62, 139 (1999). In contrast to the ubiquitous,high-level expression in most normal tissues, TSLC1 mRNA in A549 NSCLCcells was shown to be reduced to less than 15% of that seen in normallung (Example 3). Analysis of eleven additional human lungadenocarcinoma cell lines (demonstrated that TSLC1 expression was absentfrom four of the eleven. A549 and all four of the cell lines lackingTSLC1 expression form tumors in nude mice. TSLC1 expression was alsoabsent from 3 of 8 hepatocelluar carcinoma (HCC) cell lines and 8 of 11pancreatic cancer (PAC) cell lines, suggesting that it may be involvedin multiple human cancers (data not shown).

Transfected cell lines and a control A549 containing only plasmid DNAwere examined for tumorigenicity by injecting 10⁵ cells subcutaneouslyinto BALB/c athymic nu/nu mice (See FIG. 1B; Example 4). A549 andAΔTSLC1 cells both formed tumors at 8 of 8 sites of injection within 21days (FIG. 1E). These tumors continued to grow until the experiment wasterminated at 53 days. In contrast, only four of 24 injection sites ofATSLC1, 2 or 3 cell lines had palpable tumors at 21 days. Those tumorsthat eventually formed from ATSLC cells at 11 of 24 injection sites grewsubstantially slower than those of A549 to AΔTSLC1 cells. These resultsshow that TSLC1 by itself has significant tumor suppressor activity.

To determine whether LOH in human NSCLC tumors uncovers a geneticalteration in the remaining TSLC1 allele, exons 1 to 10, together withtheir flanking sequences, were examined in 12 NSCLC cell lines and 54primary NSCLC tumors using single strand conformation polymorphism(SSCP) analysis (Example 4). This analysis was also performed on 8 HCCcell lines, 36 primary HCCs, 11 pancreatic cancer cell lines and 40primary cancers from exocrine pancreas. One of the NSCLC tumors showed amobility shift accompanied by loss of wild-type fragment, while noalteration was observed in non-cancerous lung from the same patient.Sequence analysis revealed a 2-bp deletion in codons 243 to 244 of thistumor, resulting in a frameshift that is predicted to replace 19 aminoacid residues at the COOH terminus of TSLC1 with a 52-residue sequence.(FIG. 2A). A nonsense mutation in codon 208 accompanied by loss of thewild-type allele was detected in one advanced HCC, H13C (FIG. 2C). Apancreatic cancer, PC22C, also carries a cancer specific mutationchanging methionine to threonine at residue 383 in the transmembranedomain (data not shown).

Thus mutational inactivation of TSLC1 uncovered by LOH occurs in a smallsubset of primary human tumors. However, the remaining 158 tumor samplesshowed no evidence of sequence alteration in the TSLC1 gene, althoughLOH on 11q23.2 was observed in 42%, 29% and 17% of primary NSCLC, HCCand PAC, respectively (Example 5).

Given the absence of structural alteration from most TSLC1 allelesuncovered by LOH, and reduction or absence of its expression in a numberof tumorigenic cell lines, the possibility was examined that TSLC1expression is down regulated through hypermethylation of the promoter,as observed in some other genes (T. Sakai et al., Am J. Hum. Genet. 48,880 (1991); A Merlo et al, Nature Med. 1, 686 (1995); and M. F. Kane etal., Cancer Res. 57, 808 (1997)19-21). Determination of the nucleotidesequence in the region upstream from TSLC1 identified two CpG islands.Bisulfite sequencing was used to determine the methylation status of sixCpG sites in a 93-bp fragment within a CpG island containing putativepromoter sequences (Example 6). As shown in FIG. 3D, all cytosineresidues in normal lung DNA were unmethylated and therefore were alteredto thymine residues after bisulfite sequencing. In contrast, three ofthese CpG sites were methylated in the cell lines that showed completeloss of TSLC1 expression. In all, promoter sequences were methylated in4 of 4 NSCLC cell lines with loss of TSLC1 expression but not methylatedin 8 of 8 lines that essentially expressed TSLC1 (FIG. 3E). Thus,hypermethylation may act as the “second hit” to inactivate the TSLC1allele in the significant fraction of NSCLC cell lines (and tumors) thathave undergone LOH. It is interesting that methylation does not occur inthe promoters of either A549 or A431. Both show very low, albeit clearlydetectable TSLC1 mRNA levels. This suggests that further mechanismsregulating TSLC1 may be important in certain NSCLC.

Inhibition of tumor growth in inoperable patients is one of theimportant issues for the control of the disease. Loss of chromosome11q23.2 occurs in about 40% of NSCLC, deleting one allele of TSLC1.Expression of tumor suppressor gene TSLC1 is further reduced or lost bymutational inactivation or promoter methylation in a significantfraction of NSCLC and also HCC and PAC cell lines. Although the A549cell line, like advanced cancers, is known to carry multiple geneticalterations (Y. Murakami, T. Sekiya Mutation Res. 400, 421 (1998)),restoration of expression of this single gene was sufficient tosignificantly suppress the malignant phenotype of the cells. Therefore,TSLC1 and its effectors represent molecular targets for treatment ofNSCLC and other human tumors.

The following are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

EXAMPLE 1

Functional mapping of tumor suppressor activity on 11q23.2 wasaccomplished by introduction of a 1.6 MB YAC and nested deletionderivatives of it into human NSCLS tumor cell line A549. Sixty-sevenhuman ESTs from 11q23 (National Center for Biotechnology Information)were screened for tumor suppressor activity in A549 and an EST clone,SHGC-31226, was mapped within the 100 kb fragment identified on y939b12(CITB) as responsible for essentially full repression of NSCLS tumor(See FIG. 1A). TSLC1 was localized to a 100 kb segment responsibleessential for full suppression. A full-length cDNA was cloned based oncDNA sequencing, overlapping of expressed sequence tag (EST) sequencesfrom the NCBI public database found on the worldwide web atncbi.nlm.nih.gov/Entrez/, and 5′ rapid amplification of cDNA ends (RACE)using the MARATHON® cDNA Amplification Kit (Clontech) according to themanufacturer's instructions. Olgonucleotide primers used for the firstPCR reaction were as follows:

5′-CCATCCTAATACGACTCACTATAGGGC-3′  (SEQ ID NO:3)

and

5′-TCGCAACCTCTCCCTCGATCACTGTCA-3′  (SEQ ID NO:4)

and the primers used for the second PCR reaction were as follows:

5′-ACTCACTATAGGGCTCGAGCGGC-3′  (SEQ ID NO:5)

and

5′-AGAGCAACAGCAGAAGCCGGAGCCGGA-3′  (SEQ ID NO:6)

EXAMPLE 2

The subcellular localization of TSLC1 was examined by expressing aTSLC1:gfp fusion protein in COS 7 cells. A whole or a portion of codingsequences of TSLC1 were amplified by RT-PCR using adult human lungpoly-A RNA (Clontech). The following primers used for amplification ofTSLC1:

5′-GGGGTACCCAGGTGCCCGACATGGC-3′  (SEQ ID NO:7)

and

5′-AAGGAAAAAAGCGGCCGCCAGTTGGACACCTCATTGAA-3′  (SEQ ID NO:8)

The following primers were used for amplification of ΔTSLC1:

5′-TTGGTACCCGAGCTCGGATCCTCCTGGTCCCACCACGT-3′  (SEQ ID NO:9)

and

5′-AAGGAAAAAAGCGGCCGCCAGTTGGACACCTCATTGAA-3′  (SEQ ID NO:10)

Amplified fragments of TSLC1 and ΔTSLC1 were digested with restrictionendonucleases, KpnI and NotI, subcloned into plasmid pcDNA3.1-Hygro (+)(Invitrogen) to yield plasmids pcTSLC1 and pcΔTSLC1, respectively. Theinserts of pcTSLC1 and pcΔTSLC1 were then subcloned into plasmidpEGFP-N3 (Clontech), which contains a nucleotide encoding the greenfluorescent peptide (gfp) to obtain plasmids pTSLC1-GFP and pΔTSLC1-GFP.COS 7 cells from RIKEN Cell Bank, Japan, were transfected withpTSLC1-GFP or pΔTSLC1-GFP and cultured on a cover slip. Cells were fixedwith 2% paraformaldeyde 24 to 40 h after transfection and analyzed byfluorescence microscopy as described by K. Ghosh, H. P. Ghosh (Biochem.Cell Biol. 77, 165 (1999). The results of the fluorescence microscopystudies showed that TSLC1 localized in perinuclear and plasma membranes.The corresponding ΔTSLC1:gfp protein construct, lacking the signalpeptide, was expressed but failed to localize in plasma membrane.

EXAMPLE 3

Levels of expression of TSLC1 in tumor cells were determined bymeasurement of mRNA in A549 cells. Human multiple tissue Northern blotand adult lung poly-A RNA were obtained from Clontech. Poly-A RNA fromlung cancer cell lines and their derivatives was extracted using theFASTTRACK® 2.0 kit (Invitrogen). PCR utilizing primers

 5′-CATCACAGTCCTGGTCCCACCACGTAATCT-3′  (SEQ ID NO:11)

and

5′-AATAGGGCCAGTTGGACACCTCATTGAAAC-3′  (SEQ ID NO:12)

was used to derive TSLC1. Intensity of the signals was quantified usingthe BAS-2000 Imaging System (Fuji).

Northern Blot analysis of eleven additional human lung adenocarcinomacell lines (Lane 1, A549; lane 2, ABC-1; lane 3, Calu-3; lane 4,NCI-H441; lane 5 NCI-H522; lane 6, LCMS; lane 7, LCOK; lane 8, VMRC-LCD;lane 9, PC-14; lane 10, SK-LU-1; lane 11, NCI-H596; lane 12, A431; lane13, normal lung) showed that TSLC1 expression was absent from 4 of the11 additional cell lines.

Similar Northern blot analysis found TSLC1 expression missing from 3 of8 hepatocellular carcinoma cell lines (HCC) and 8 of 11 pancreaticcancer cell lines.

Expression of TSLC1 in various healthy tissues was studied by Northernblot analysis in healthy human heart; brain; placenta; lung; liver;skeletal muscle; kidney; and pancreas tissues. β-actin expression wasused as a control. Strong expression of TSLC1 was found in healthyheart, brain, placenta and lung (lanes 1-4) with moderate expression inskeletal muscle, kidney, and pancreas (lanes 6-8).

The study was repeated in several NSCLC lines using Northern blotanalysis of TSLC1 mRNA, with normal lung and β-actin as controls. TSLC1mRNA was present in ABC-1, NCI-H522 and VMRC-LCD cell lines and innormal lung, but was reduced or absent in several other NSCLC celllines. Expression was completely absent in PC-14, SK-LU-1, and NCI-H596cell lines (lanes 9-11).

EXAMPLE 4

To determine whether LOH in human NSCLC tumors uncovers a geneticalteration in the remaining TSLC1 allele, exons 1 to 10, together withtheir flanking sequences, were examined in 12 NSCLC cell lines and 54primary NSCLC tumors using single strand conformation polymorphism(SSCP) analysis. Primary tumors were supplied by Pathology Division,National Cancer Center Research Institute and Japan Research Group ofPancreatic Cancer. All the experiments using human materials wereperformed in accordance with the institutional guidelines. Genomic DNAwas extracted by Proteinase K-phenol-chloroform extraction method.PCR-SSCP analysis was essentially carried out using a method asdescribed previously by M. Orita et al. (Genomics 5, 874 (1989)), whichis incorporated herein by reference in its entirety.

Primer pairs used for amplification of exon 5 of TSLC1 were as follows:

5′-CACCCAACTCTGGTGTCTTGGTAC-3′  (SEQ ID NO:13)

and

5′-CTCTACGCCCTCAGAATAAGATAC-3′  (SEQ ID NO:14).

Primer pairs used for amplification of exon 10 of TSLC1 were as follows:

5′-TTACACAGAGGCCATCAGACAGTC-3′  (SEQ ID NO:15)

and

5′-AAATAGGGCCAGTTGGACACCTC-3′  (SEQ ID NO: 16).

Amplification products were denatured at 95° C. for 3 min and separatedon 5% polyacrylamide gels with buffer containing2-[N-morpholino]ethanesulfonic acid as described in Y. Kukita et al.(Hum. Mutation 10, 400 (1997). SSCP analysis of exons 5 and 10 of TSLC1in cell lines L213N, L255C, L255N, L213N, H13C and H13N showed loss ofthe wild type allele and mobility shift in the remaining allele in L255Cand H13C. One NSCLC tumor showed a mobility shift accompanied by loss ofwild-type fragment, while no alteration was observed in non-cancerouslung from the same patient.

DNA fragments were eluted from gels, amplifed, and sequenced by the ABIPRISM® dye terminator cycle sequencing ready reaction kit (Perkin-Elmer)using an ABI 377 DNA auto-sequencer (Applied Biosystems). Sequenceanalysis revealed a 2-bp deletion in codons 243 to 244 of this tumor,resulting in a frameshift that is predicted to replace 19 amino acidresidues at the COOH terminus of TSLC1 with a 52-residue sequence.

EXAMPLE 5

Human multiple tissue Northern blot and adult lung poly-A RNA wereobtained from Clontech. Poly-A RNA from lung cancer cell lines and theirderivatives was extracted using the FASTTRACK® 2.0 kit (Invitrogen). A961-bp PCR-derived fragment of SEQ ID NO:17 (nt 411-1,371 of TSLC1 cDNA)obtained using primers

5′-CATCACAGTCCTGGTCCCACCACGTAATCT-3′  (SEQ ID NO:18)

and

5′-AATAGGGCCAGTTGGACACCTCATTGAAAC-3′  (SEQ ID NO:19)

was used as a probe for detection of TSLC1 in lung cancer cell lines.Intensity of the signals was quantified using the BAS-2000 (Fuji ImagingSystem).

EXAMPLE 6

To examine tumor suppressor activity of the TSLC1 gene, mini-genes wereconstructed that carry the complete coding sequence of TSLC1 or atruncated version lacking the NH2-terminal signal peptide (ΔTSLC1).TSLC1 expression was restored by transfection of mini-genes into A549.Plasmids pcTSLC1, pcΔTSLC1 and pcDNA3.1-Hygro(+) were transfected intoA549 cells using LIPOFECTAMINE PLUS® (GIBCO BRL) and hygromycinresistant cells were cloned to derive three independent lines containingfull length TSLC1 (ATSLC1, 2, 3) and one cell line with the truncatedmini-gene (AΔTSLC1). Northern blot analysis showed relative levels ofTSLC1 transcripts of 4.4 kb and 1.6 kb, transcripts from full length(TSLC1, 1.8 kb) and truncated (ATSLC1, 1.4 kb) mini-genes, and β-actin(as control). These results showed that the mini-genes encoding fulllength TSLC1 restored TSLC1 mRNA levels in A549 from 15% of thatobserved in normal lung tissue to levels 120%, 660% and 100% of thatobserved in normal lung tissue in ATSLC1, 2, and 3, respectively.AΔTSLC1 expressed the truncated message at levels similar to ATSLC1 and3. As reported above, in AΔTSLC1 this message is translated but theresulting protein does not localize to the cell membrane. This resultindicates that the tumor suppressor activity of TSLC1 depends uponlocalization of the encoded protein to the cell membrane.

EXAMPLE 7

Transfected cell lines AΔTSLC1, 2 and 3 (which each contain a promoterplus a TSLC1 cDNA that lacks the N-terminal signal sequences) and acontrol A549 containing only plasmid DNA were examined fortumorigenicity by injecting cells of each subcutaneously into BALB/cathymic nu/nu mice. A suspension of 1×10⁵ cells in 0.2 ml PBS wasinjected subcutaneously into one to four sites on the flanks of 5- to6-week old female BALB/c athymic nu/nu mice (Charles River). Tumorgrowth was assessed by measuring the xenografts in three dimensions. Allanimal experiments were performed in accordance with the institutionalguidelines.

In these experiments A549 and AΔTSLC1 cells both formed tumors at 8 of 8sites of injection within 21 days (FIG. 1E). These tumors continued togrow until the experiment was terminated at 53 days. In contrast, onlyfour of 24 injection sites of ATSLC1, 2, or 3 cell lines (which containa promoter plus a full length TSLC1 cDNA) had palpable tumors at 21days. Those tumors that eventually formed from ATSLC cells at 11 of 24injection sites grew substantially slower than those of A549 or AΔTSLC1cells. These experiments show that TSLC1 by itself has significant tumorsuppressor activity.

EXAMPLE 8

DNA fragments containing four polymorphic STS markers, D11S4111,D11S1235, D11S2077 and D11S1885, were amplified by PCR from NSCLC, HCCand PAC tumors and non-cancerous tissues of the same patients usingpairs of primers, one of which was labeled with ³²P-dCTP. Amplifiedfragments were subjected to electrophoresis in polyacrylamide gelscontaining 7M Urea and autoradiography. Loss of heterozygosity on11q23.2 was observed in 42%, 29% and 17% of primary HSCLC, HCC AND PAC,respectively.

EXAMPLE 9

To determine whether TSLC1 is down regulated through hypermethylation ofthe promoter, the nucleotide sequence in the region upstream from TSLC1was examined to identify two CpG islands. Bisulfite sequence was used todetermine the methylation status of six CpG sites in a 93-bp fragmentwithin a CpG island containing putative promoter sequences as describedby M. Frommer et al. (Proc. Natl. Acad. Sci. USA 89, 1827 (1992). Afterdenaturing with NaOH (0.3M), genomic DNA (2 μg) was incubated withsodium bisulfite (3.1 M; Sigma) and hydroquinone (0.8 mM; Sigma) pH 5.0,at 55° C. for 20 h, purified and treated with NaOH (0.2 M) for 10 min at37° C. Modified DNA (100 ng) was subjected to PCR to amplify thepromoter sequence of TSLC1 with the following primers:

5′-GTGAGTGACGGAAATTTGTAATGTTTGGTT-3′  (SEQ ID NO:20)

and

5′-AATCTAACTTCTTATACACCTTTATTAAAA-3′  (SEQ ID NO:21).

The PCR products were purified and directly sequenced to obtain averagemethylation levels. PCR products containing bisulfite-resistantcytosines were subcloned and at least 6 clones were sequenced forconfirmation (FIGS. 2D and 2E).

EXAMPLE 10

Bisulfite sequencing was used to determine the methylation status of sixCpG sites in a 93-bp fragment within a CpG island containing putativepromoter sequences. Bisulfite sequencing was performed as described (M.Frommer et al., Proc. Natl. Acad. Sci. USA 89, 1827 (1992)). Afterdenaturing with NaOH (0.3M), genomic DNA (2 μg) was incubated withsodium bisulfite (3.1 M; Sigma) and hydroquinone (0.8 mM; Sigma) pH 5.0,at 55° C. for 20 h, purified and treated with NaOH (0.2 M) for 10 min at37° C. Modified DNA (100 ng) was subjected to PCR to amplify thepromoter sequence of TSLC1 with the following primers:

5′-GTGAGTGACGGAAATTTGTAATGTTTGGTT-3′  (SEQ ID NO:22

and

5′-AATCTAACTTCTTATACACCTTTATTAAAA-3′  (SEQ ID NO:23).

The PCR products were purified and directly sequenced to obtain averagemethylation levels. PCR products containing bisulfite-resistantcytosines were subcloned and at least 6 clones were sequenced forconfirmation. The results of these studies are shown in FIGS. 2A-E.

It will be apparent to those skilled in the art that various changes maybe made in the invention without departing from the spirit and scopethereof, and therefore, the invention encompasses embodiments inaddition to those specifically disclosed in the specification, but onlyas indicated in the appended claims.

32 1 442 PRT Homo sapiens 1 Met Ala Ser Val Val Leu Pro Ser Gly Ser GlnCys Ala Ala Ala Ala 1 5 10 15 Ala Ala Ala Ala Pro Pro Gly Leu Arg LeuArg Leu Leu Leu Leu Leu 20 25 30 Phe Ser Ala Ala Ala Leu Ile Pro Thr GlyAsp Gly Gln Asn Leu Phe 35 40 45 Thr Lys Asp Val Thr Val Ile Glu Gly GluVal Ala Thr Ile Ser Cys 50 55 60 Gln Val Asn Lys Ser Asp Asp Ser Val IleGln Leu Leu Asn Pro Asn 65 70 75 80 Arg Gln Thr Ile Tyr Phe Arg Asp PheArg Pro Leu Lys Asp Ser Arg 85 90 95 Phe Gln Leu Leu Asn Phe Ser Ser SerGlu Leu Lys Val Ser Leu Thr 100 105 110 Asn Val Ser Ile Ser Asp Glu GlyArg Tyr Phe Cys Gln Leu Tyr Thr 115 120 125 Asp Pro Pro Gln Glu Ser TyrThr Thr Ile Thr Val Leu Val Pro Pro 130 135 140 Arg Asn Leu Met Ile AspIle Gln Lys Asp Thr Ala Val Glu Gly Glu 145 150 155 160 Glu Ile Glu ValAsn Cys Thr Ala Met Ala Ser Lys Pro Ala Thr Thr 165 170 175 Ile Arg TrpPhe Lys Gly Asn Thr Glu Leu Lys Gly Lys Ser Glu Val 180 185 190 Glu GluTrp Ser Asp Met Tyr Thr Val Thr Ser Gln Leu Met Leu Lys 195 200 205 ValHis Lys Glu Asp Asp Gly Val Pro Val Ile Cys Gln Val Glu His 210 215 220Pro Ala Val Thr Gly Asn Leu Gln Thr Gln Arg Tyr Leu Glu Val Gln 225 230235 240 Tyr Lys Pro Gln Val His Ile Gln Met Thr Tyr Pro Leu Gln Gly Leu245 250 255 Thr Arg Glu Gly Asp Ala Leu Glu Leu Thr Cys Glu Ala Ile GlyLys 260 265 270 Pro Gln Pro Val Met Val Thr Trp Val Arg Val Asp Asp GluMet Pro 275 280 285 Gln His Ala Val Leu Ser Gly Pro Asn Leu Phe Ile AsnAsn Leu Asn 290 295 300 Lys Thr Asp Asn Gly Thr Tyr Arg Cys Glu Ala SerAsn Ile Val Gly 305 310 315 320 Lys Ala His Ser Asp Tyr Met Leu Tyr ValTyr Asp Pro Pro Thr Thr 325 330 335 Ile Pro Pro Pro Thr Thr Thr Thr ThrThr Thr Thr Thr Thr Thr Thr 340 345 350 Thr Ile Leu Thr Ile Ile Thr AspSer Arg Ala Gly Glu Glu Gly Ser 355 360 365 Ile Arg Ala Val Asp His AlaVal Ile Gly Gly Val Val Ala Val Val 370 375 380 Val Phe Ala Met Leu CysLeu Leu Ile Ile Leu Gly Arg Tyr Phe Ala 385 390 395 400 Arg His Lys GlyThr Tyr Phe Thr His Glu Ala Lys Gly Ala Asp Asp 405 410 415 Ala Ala AspAla Asp Thr Ala Ile Ile Asn Ala Glu Gly Gly Gln Asn 420 425 430 Asn SerGlu Glu Lys Lys Glu Tyr Phe Ile 435 440 2 1329 DNA Homo sapiensmisc_feature (1)..(1329) n is any nucleotide 2 tatcgcagag gttaagntggagtggtgcnt ttggaacccc cggatccccc tttgtctcac 60 ccaccccgnt ntnttnttccaattgttttn tccccttntg ngcntgnaac cgagttnggg 120 ntgatgatgn ccataaagcaagttgccatc tctgtaccac tttacacaga ggccatcaga 180 cagtcacggt gctttaccccttcatctttc aggtacatac ttcactcatg aagccaaagg 240 agccgatgac gcagcagacgcagacacagc tataatcaat gcagaaggag gacagaacaa 300 ctccgaagaa aagaaagagtacttcatcta gatcagcctt tttgtttcaa tgaggtgtcc 360 aactggccct atttagatgataaagagaca gtgatattgg aacttgcgag aaattcgtgt 420 gtttttttat gaatgggtggaaaggtgtga gactgggaag gcttgggatt tgctgtgtaa 480 aaaaaaaaaa aaaatgttctttggaaagta cactctgctg tttgacacct cttttttcgt 540 ttgtttgttt gtttaatttttatttctncc taccaagtca aacttggata cttggattta 600 gtttcagtag attgcagaaaattctgtgcc ttgttttttg tttgtttgtt gcgtnccttt 660 cttttccccc tttgtgcacatttatttcct ccctctaccc caatttcgga ttttttccaa 720 aatctcccat tttggaatttgcctgctggg attccttaga ctcttttcct tcccttttct 780 gttctagttt tttacttttgtttattttta tggtaactgc tttctgttcc aaattcagtt 840 tcataaaagg agaaccagcacagcttagat ttcatagttc agaatttagt gtatccataa 900 tgcattcttc tctgttgtcgtaaagatttg ggtgaacaaa caatgaaaac tctttgctgc 960 tgcccatgtt tcaaatacttagagcagtga agactagaaa attagactgt gattcagaaa 1020 atgttctgtt tgctgtggaactacattact gtacagggtt atctgcaagt gaggtgtgtc 1080 acaatgagat tgaatttcactgtctttaat tctgtatctg tagacggctc agtatagata 1140 ccctacgctg tccagaaaggtttggggcag aaaggactcc tcctttttcc atgccctaaa 1200 cagacctgac aggtgaggtctgttcctttt atataagtgg acaaattttg agttgccaca 1260 ggaggggaag tagggaggggggaaatacag ttctgctctg gttgtttctg ttccaaatga 1320 ttccatcca 1329 3 27 DNAArtificial sequence Primer for PCR 3 ccatcctaat acgactcact atagggc 27 427 DNA Artificial sequence Primer for PCR 4 tcgcaacctc tccctcgatcactgtca 27 5 23 DNA Artificial sequence Primer for PCR 5 actcactatagggctcgagc ggc 23 6 27 DNA Artificial sequence Primer for PCR 6agagcaacag cagaagccgg agccgga 27 7 25 DNA Artificial sequence Primer forPCR 7 ggggtaccca ggtgcccgac atggc 25 8 38 DNA Artificial sequence Primerfor PCR 8 aaggaaaaaa gcggccgcca gttggacacc tcattgaa 38 9 38 DNAArtificial sequence Primer for PCR 9 ttggtacccg agctcggatc ctcctggtcccaccacgt 38 10 38 DNA Artificial sequence Primer for PCR 10 aaggaaaaaagcggccgcca gttggacacc tcattgaa 38 11 30 DNA Artificial sequence PCRutilizing primer 11 catcacagtc ctggtcccac cacgtaatct 30 12 30 DNAArtificial sequence PCR utilizing primer 12 aatagggcca gttggacacctcattgaaac 30 13 24 DNA Artificial sequence Primer for PCR 13 cacccaactctggtgtcttg gtac 24 14 24 DNA Artificial sequence Primer for PCR 14ctctacgccc tcagaataag atac 24 15 24 DNA Artificial sequence Primer forPCR 15 ttacacagag gccatcagac agtc 24 16 23 DNA Artificial sequencePrimer for PCR 16 aaatagggcc agttggacac ctc 23 17 961 DNA Homo sapiensmisc_feature (1)..(961) n is any nucleotide 17 gacagtgata ttggaacttgcgagaaattc gtgtgttttt ttatgaatgg gtggaaaggt 60 gtgagactgg gaaggcttgggatttgctgt gtaaaaaaaa aaaaaaaatg ttctttggaa 120 agtacactct gctgtttgacacctcttttt tcgtttgttt gtttgtttaa tttttatttc 180 tncctaccaa gtcaaacttggatacttgga tttagtttca gtagattgca gaaaattctg 240 tgccttgttt tttgtttgtttgttgcgtnc ctttcttttc cccctttgtg cacatttatt 300 tcctccctct accccaatttcggatttttt ccaaaatctc ccattttgga atttgcctgc 360 tgggattcct tagactcttttccttccctt ttctgttcta gttttttact tttgtttatt 420 tttatggtaa ctgctttctgttccaaattc agtttcataa aaggagaacc agcacagctt 480 agatttcata gttcagaatttagtgtatcc ataatgcatt cttctctgtt gtcgtaaaga 540 tttgggtgaa caaacaatgaaaactctttg ctgctgccca tgtttcaaat acttagagca 600 gtgaagacta gaaaattagactgtgattca gaaaatgttc tgtttgctgt ggaactacat 660 tactgtacag ggttatctgcaagtgaggtg tgtcacaatg agattgaatt tcactgtctt 720 taattctgta tctgtagacggctcagtata gataccctac gctgtccaga aaggtttggg 780 gcagaaagga ctcctcctttttccatgccc taaacagacc tgacaggtga ggtctgttcc 840 ttttatataa gtggacaaattttgagttgc cacaggaggg gaagtaggga ggggggaaat 900 acagttctgc tctggttgtttctgttccaa atgattccat ccacctttcc caatcggcct 960 t 961 18 30 DNAArtificial sequence Primer for PCR 18 catcacagtc ctggtcccac cacgtaatct30 19 30 DNA Artificial sequence Primer for PCR 19 aatagggcca gttggacacctcattgaaac 30 20 30 DNA Artificial sequence Primer for PCR 20 gtgagtgacggaaatttgta atgtttggtt 30 21 30 DNA Artificial sequence Primer for PCR 21aatctaactt cttatacacc tttattaaaa 30 22 30 DNA Artificial sequence Primerfor PCR 22 gtgagtgacg gaaatttgta atgtttggtt 30 23 30 DNA Artificialsequence Primer for PCR 23 aatctaactt cttatacacc tttattaaaa 30 24 153DNA Homo sapiens 24 gtgagtgacg gaaatttgca acgtctggtt cgctaggccagatgcactcg gtgtgcggga 60 cagaggaccc tcttaaggga gattctccag tcgtcggtctgatacagcga ttgctataaa 120 cattcctaat aaaggtgtac aagaagctag acc 153 25 18DNA Homo sapiens 25 tgttggtttg atatagtg 18 26 18 DNA Homo sapiens 26cgtcggtttg atatagcg 18 27 18 DNA Homo sapiens 27 cgtcggtttg atatagcg 1828 18 DNA Homo sapiens 28 tgttggtttg atatagtg 18 29 18 DNA Homo sapiens29 acacagctat aatcaatg 18 30 18 DNA Homo sapiens 30 acacagctaa tcaatgca18 31 16 DNA Homo sapiens 31 tgatgctgaa ggtgca 16 32 16 DNA Homo sapiens32 tgatgctgta ggtgca 16

What is claimed is:
 1. A method of detecting cancer associated withtumor suppressor lung cancer 1 (TSLC1) in a subject in need thereof,said method comprising contacting a nucleic acid sample of the subjectwith a reagent that detects the methylation status of the TSLC1 promoterand determining the status as compared with a comparable healthy cell,wherein the difference in methylation status between the sample from thesubject and the healthy cell is indicative of cancer in the subject. 2.The method of claim 1, wherein the reagent is a restrictionendonuclease.
 3. The method of claim 2, wherein the restrictionendonuclease is methylation sensitive.
 4. A method of detecting canceror predisposition to cancer in a subject in need thereof, said methodcomprising contacting nucleic acid of a test cell with a reagent thatdetects the methylation status of the TSLC1 promoter and detecting adifference in methylation status in the TSLC1 promoter as compared tothat of a comparable normal cell, wherein the difference in methylationstatus between the sample from the subject and the healthy cell isindicative of cancer in the subject; wherein the cancer is lung, liveror pancreatic cancer.
 5. The method of claim 4, wherein the lung canceris human non-small cell lung cancer.
 6. The method of claim 4, whereinthe liver cancer is hepatocellular carcinoma.
 7. The method of claim 4,wherein said detecting determines increased methylation of the TSLC1promoter in the test cell.
 8. The method of claim 4, wherein subject hasloss of heterozygosity of 11q23 chromosome.
 9. The method of claim 4,wherein the difference in methylation results in reduced production ofTSLC1 in the test cell as compared to the comparable normal cell. 10.The method of claim 1, wherein the cancer is lung, liver or pancreaticcancer.
 11. The method of claim 10, wherein the lung cancer is humannon-small cell lung cancer.
 12. The method of claim 10, wherein theliver cancer is hepatocellular carcinoma.
 13. The method of claim 10,wherein the liver cancer is hepatocellular carcinoma.
 14. The method ofclaim 1 or 4, wherein the methylation status is determined by bisulfitesequencing.